Production of itaconic acid by fermentation

ABSTRACT

Itaconic acid and/or salt thereof is produced via aerobic microbial fermentation, for example by means of the species Aspergillus terreus or Aspergillus itaconicus, of a nutrient medium containing a source of assimilable carbon, such carbon source at least in part comprising an effective amount of glycerol.

This application is a divisional of application Ser. No. 08/205,646,filed Mar. 4, 1994 now U.S. Pat. No. 5,457,040.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the preparation of itaconic acid byfermentation, and, more especially, via microbial fermentation ofparticular nutrient media.

2. Description of the Prior Art

The production of organic acids by fermentation of sugars in thepresence of a suitable microorganism is universally known to this art.Typical acids of such fermentations include, in particular, acetic acid,lactic acid, citric acid, fumaric acid and, more especially, itaconicacid.

Itaconic acid is a dicarboxylic acid having the following structuralformula: ##STR1##

By reason of its site of ethylenic unsaturation, itaconic acid esterspolymerize very readily and are thus widely used in the detergent andsynthetic plastics industries and in the preparation of adhesives.

Traditionally, the production of itaconic acid is carried out byfermentation. Various varieties of the genus Aspergillus, and moreespecially those of Aspergillus terreus and Aspergillus itaconicus, areused for producing itaconic acid via fermentation of carbohydrates.

The carbohydrates most typically used include mono- and disaccharides,such as glucose, sucrose and fructose and starches, as they exist in aform which is assimilable by the microorganism, and molasses.

Indeed, in traditional fermentations, from an economic standpoint, theselection of the hydrocarbon substrate should be based both on its cost,on its availability and on its capacity to provide high yields.

Thus, in order to economically optimize the processes for preparingitaconic acid, it is necessary to select a hydrocarbon source thatsimultaneously satisfies the aforesaid three requirements.

SUMMARY OF THE INVENTION

Accordingly, a major object of the present invention is the provision ofan improved fermentation process, utilizing a unique carbon source whichis available in large amounts and at lower cost and which is completelyassimilable by the microorganisms suitable for the production ofitaconic acid.

Briefly, the present invention features a process for the production ofitaconic acid and/or the salts thereof by aerobic fermentation of acarbohydrate via suitable microorganism, and wherein the carbonsubstrate or source of the nutrient medium at least in part comprises aneffective amount of glycerol.

DETAILED DESCRIPTION OF BEST MODE AND PREFERRED EMBODIMENTS OF THEINVENTION

More particularly according to the present invention, it has nowunexpectedly been determined that glycerol is fully assimilable bymicroorganisms that are suitable for the fermentative production ofitaconic acid. In comparison with the other carbon substrates indicatedabove, glycerol presents the distinct advantage of being especiallyadvantageous from an economic standpoint. It is readily available invast amounts and at very low cost. Its use as the carbon substrate orsource provides high yields of itaconic acid at fermentation timescomparable to those employed with the traditional carbon substrates.

It will of course be appreciated that the glycerol may be used eitheralone or mixed with at least one other carbohydrate. The latter may beselected from among, for example, the mono- and disaccharides such asglucose, sucrose and fructose, starches and molasses.

In mixtures of this type, the respective proportions of glycerol andadditional carbohydrate(s) can vary widely, with the proviso that theoverall amount thereof is sufficient to permit the fermentation processto proceed effectively.

For the formulation of the corresponding nutrient media, it ispreferable to employ a concentration of carbon substrate, whetherglycerol alone or mixed with one or more other additional carbohydrates,ranging from 10 to 240 g/l expressed in terms of weight/volume.

In one particular embodiment of the invention, the itaconic acid ispartially neutralized during the fermentation process, by the addition,for example, of alkali of the sodium hydroxide or potassium hydroxidetype. In this particular case, the itaconic acid is obtained mixed withone of its inorganic salts, for example the sodium or potassium salt.

The glycerol may be introduced into the nutrient medium in several ways.Traditionally, it is introduced directly, whether or not with anothercarbohydrate, into the nutrient medium. However, the glycerol canalready be present as the primary carbon substrate in the preparation ofthe inoculum, or of the preculture of the selected microorganism. Thisinoculum is transferred in a subsequent step into a nutrient medium toinitiate the fermentation. Given this option, the concentration ofglycerol, whether or not mixed with another carbohydrate, issupplemented in the nutrient medium to attain the requisiteconcentration described above.

Particularly exemplary for carrying out the fermentation process of theinvention include the species Aspergillus terreus and Aspergillusitaconicus.

Preferred is the species Aspergillus terreus, and preferably the strainNRRL 1960 identified in EP-341,112.

Other than the glycerol according to the invention, the nutrient mediumalso contains other nutrient ingredients required for fermentation. Oneskilled in this art can easily select these other ingredients and therespective amounts thereof.

The nitrogen source can, in particular, be selected from amongmetabolizable organic or inorganic compounds, such as soluble extract ofmaize (SCL) and/or of soya bean, urea, ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium nitrate and the like, andmixtures thereof.

The medium contains, in addition, inorganic salts such as Ca, Mg, Na, K,Fe, Ni, Co, Cu, Mn, Zn sulfates, chlorides and phosphates, as well asother common additives such as pH regulators and/or antifoams.

The microorganism is itself introduced into the fermentation medium inconventional manner, by means of inoculum or of intermediate cultures.

The fermentation is suitably carried out at an acid pH ranging fromapproximately 1.8 to 5 and at a temperature of approximately 20° toapproximately 40° C., optimal conditions depending on the particularstrain of the microorganism employed.

In order to further illustrate the present invention and the advantagesthereof, the following specific example is given, it being understoodthat same are intended only as illustrative and in nowise limitative.

EXAMPLE

The production of itaconic acid was carried out using several nutrientmedia that differed from one another in the amount of glycerol employed.

The nutrient solution used contained 0.5 g of maize extract (CSL), 1.2 gof ammonium nitrate, 0.3 g of hydrated magnesium sulfate, 0.3 g ofmagnesium oxide, 0.315 g of calcium hydroxide, 0.05 g of monopotassiumphosphate and 0.380 g of hydrated copper nitrate. The total volume ofthis production medium was adjusted to 1 liter with water, and its pHwas adjusted to a value on the order of 2.8 to 3 by means of nitric acidsolution.

Depending on the test carried out, this nutrient medium was supplementedwith glycerol, mixed or otherwise with sucrose. The respective amountsof each of the carbon substrates are reported in the Table below.

For each test, the above nutrient medium was introduced into a 500 mlErlenmeyer flask and inoculated with an Aspergillus terreus NRRL 1960culture such as to provide a concentration on the order of 5×10⁻⁷spores/ml in the medium. The temperature of the medium was adjusted to32°-35° C.

Fermentation was terminated after the sugar had been exhausted and whenthe acidity was at a maximum and stable.

Samples of musts were taken at the end of the fermentation to evaluatethe itaconic acid content, determined by high performance liquidchromatography.

The productivity of itaconic acid for each test is reported in the Tablebelow.

The results obtained demonstrated that glycerol was a hydrocarbonsubstrate which was fully assimilable by the microorganism Aspergillusterreus, and, hence, suitable for the production of itaconic acid insatisfactory yields.

                                      TABLE                                       __________________________________________________________________________    Carbohydrate                     Results at 161 or 233                        Content of the                                                                             Acidities           Hours                                        Media        (g/l)               Glycerol                                                                            Itaconic                                                                           Molar                             Test                                                                             Sucrose                                                                            Glycerol                                                                           72  96  120 161 233 Remaining                                                                           Acid Yld.                              No.                                                                              (g/l)                                                                              (g/l)                                                                              Hours                                                                             Hours                                                                             Hours                                                                             Hours                                                                             Hours                                                                             (g/l) (g/l)                                                                              %                                 __________________________________________________________________________    1  75   25   10.05                                                                             26   42.25                                                                            55.9                                                                               64.35                                                                            5.8   57.85                                                                              80                                2  50   50    7.15                                                                             21.45                                                                             35.6                                                                              48.75                                                                             60.5                                                                              8.7   57.15                                                                              80.1                              3  25   75   10.5                                                                              20.3                                                                              29.6                                                                              41.95                                                                             58.2                                                                              10.7  52.75                                                                              74.15                             4   0   100  10.25                                                                             20.5                                                                              31.0                                                                              46.65                                                                             55.9                                                                              2.9   49.6 62.8                              5  110   0   19.8                                                                              38   56.55                                                                            76.7                                                                              --  --    69.7 83.8                              __________________________________________________________________________

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof.

What is claimed is:
 1. An improved process for the production ofitaconic acid and/or a salt thereof by aerobic microbial fermentation ofa nutrient medium containing a source of assimilable carbon and at leastone microorganism which produces itaconic acid and/or a salt thereofwhen cultured in the presence of said assimilable carbon, theimprovement wherein said source of assimilable carbon at least in partcomprises an effective amount of glycerol and the microorganism is astrain of Aspergillus terreus or Aspergillus itaconicus.
 2. Thefermentation process of claim 1 wherein the fermentation mediumcomprises from 10 to 240 g/l of said source of assimilable carbon. 3.The fermentation process of claim 2 wherein the fermentation mediumcomprises from about 25 to 100 g/l of glycerol.
 4. The fermentationprocess of claim 2 wherein the microorganism is a strain of Aspergillusterreus.
 5. The fermentation process as defined by claim 1, said sourceof assimilable carbon also comprising a carbohydrate.
 6. Thefermentation process as defined by claim 1, the fermentation medium alsocomprising a nitrogen source and at least one inorganic salt.